1. Field of the Invention
3'-(2')-Amino- or thiol-modified, fluorescent dye-coupled nucleosides, nucleotides and oligonucleotides, and a process for the preparation and the use thereof
2. Description of the Related Art
Labeled oligonucleotides have found an extremely large number of uses in genetic engineering because they are easier to handle than the DNA probes which are conventionally used as hybridization probes and are prepared from native gene material by restriction digestion.
Labeled oligonucleotides which are employed in the form of so-called antisense DNA oligonucleotides are able to intervene in cellular events in a regulating manner and are thus becoming of increasing importance, for example for the in vivo investigation of protein expression. According to present knowledge, the mechanism takes place via DNA-DNA, DNA-RNA and RNA-RNA interactions but the details have not yet been elucidated.
Labeled oligonucleotides are used in vitro for example for the identification of gene fragments within a gene bank by using the labeled oligonucleotide to probe and identify blotted gene probes of the gene bank.
In order to be able to carry out such experiments in vitro or in vivo, the oligonucleotides must, as already mentioned, be labeled. Besides radioactive labeling by suitable isotopes, one form of non-radioactive labeling which has already been used is derivatized fluorescent dyes which offer the possibility of easier and less hazardous handling.
To date, a technique of this type has also already been used successfully for the non-radioactive sequencing of DNA. The approaches for this are essentially based on the method of Sanger (F. Sanger, S. Nicklen and S. Coulson, Proc. Natl. Acad. Sci. USA 74, 5463 (1977)).
The fluorescent label is attached (G. L. Trainor, Anal. Chem. 62, 418 (1990)) either at the 5' end of the oligonucleotide (L. E. Hood, L. M. Smith and C. Heiner, Nature 321, 674 (1986)) or at the nucleobase (J. M. Prober, G. L. Trainor and R. J. Dam, Science 238, 336 (1987)). A crucial disadvantage of the last-mentioned method derives from the fact that the fluorescent labeling is introduced during the synthesis, i.e. during the polymerization and, in this case, specifically during the enzymatic polymerization. This step in the method has the consequences that only a few polymerases can now be used for the synthesis, that the acceptance of the triphosphates by the polymerases is diminished and that, moreover, a large substrate excess is necessary.
However, the introduction of a fluorescent label is not confined to Sanger sequencing. Maxam-Gilbert chemical sequencing with a fluorescent label is also known (H. Voss, C. Schwager, U. Wirkner, B. Sproat, J. Zimmermann, A. Rosenthal, H. Erfle, J. Stegemann and W. Ansorge, Nucl. Acids Res. 17, 2517 (1989)).
In analogy to this, the mapping of restriction fragments with fluorescence detectors is also described (A. V. Carrano, J. Lamerdin, L. K. Ashworth, B. Watkins, E. Branscomb, T. Slezak, M. Raff, P. J. de Jong, D. Keith, L. McBride, S. Meister, M. Kronick, Genomics 4, 129 (1989) and S. Brenner and K. J. Livak, Proc. Natl. Acad. Sci. USA 86, 8902 (1989)).